Traditionally, the pulmonary surfactant protein-A (SP-A) is thought to opsonize and enhance the clearance of microbial pathogens. Recently, we have reported that SP-A also directly kills Pseudomonas aeruginosa (PA) in a macrophage-independent manner, by increasing the permeability of bacterial membranes. However, the mechanism by which SP-A disrupts PA cell membranes and its relative importance in lung defense are poorly defined. In addition, how microbes protect themselves against SP-A is unknown. Especially, we have shown that wild-type PA strain PA01 is resistant to membrane permeabilization by SP-A. Our long-term goal is to understand the antimicrobial mechanisms of SP-A, and to reveal how bacteria confer resistance/susceptibility to SP-A. The overall hypothesis to be tested is that PA pathways including flagellum, salicylate and pyochelin, and phosphoenol-pyruvate phosphotransferase, are important to resist membrane permeabilization by SP-A. Our hypothesis is supported by our published and preliminary data which show that PA mutant strains deficient in flagellum (flgE), deficient in salicylate and pyochelin biosynthesis (pchA), and defective in producing phosphoenol-pyruvate phosphotransferase (ptsP), are preferentially cleared in the SP-A+/+ mouse lungs, but survived in the SP-A-/- mouse lungs. Most strikingly, the flgE, pchA and ptsP mutant bacteria show significant increase in susceptibility to SP-A mediated membrane permeabilization, but not opsonization. We propose three aims to examine the mechanisms by which flagellum, PchA and PtsP pathways maintain LPS and cell membrane integrity, and regulate the bacterial processes that inactivate SP-A, to confer resistance to killing by SP-A-mediated membrane permeabilization and to killing by SP-A and antimicrobial peptides and proteins (AMPPs) whose functions that are either dependent or aided by SP-A. Aim 1 will determine the offensive strategies orchestrated by PA's flagellum, PchA and PtsP to confer resistance to SP-A-mediated membrane permeabilization. These offensive measures to be tested include the ability of PA to secrete proteases that degrade SP-A, and to secrete salicylate that strips Ca2+ associated with SP-A, which is required for the activity of SP-A. Aim 2 will determine the defensive strategies orchestrated by PA's flagellum, salicylate/pyochelin and phosphoenol-pyruvate phosphotransferase to confer resistance to SP-A-mediated membrane permeabilization. The defensive measures to be determined include increased LPS biosynthesis and modification, and chemotaxis evasion. Aim 3 will examine the hypothesis that during the initial interactions, SP-A preemptively paralyzes PA, allowing other AMPPs to act synergistically or additively to kill the bacteria. We will use checker board assays to determine the roles of flagellum, PchA and PtsP pathways against individual, synergistic and additive killing by individual AMPP alone, different combinations of AMMPs, and AMMPs with SP-A. Completion of the proposed aims will enhance our understanding of the antimicrobial mechanisms of the SP-A and lead to new treatment strategies for pneumonias. PUBLIC HEALTH RELEVANCE: Pseudomonas aeruginosa is one of the most common causes of nosocomial infections in humans, lung infections in cystic fibrosis patients, and a primary cause of death and sepsis in immuno-compromised individuals. The continuous emergence of antibiotic resistant P. aeruginosa, which can lead to denial for lung transplant, infection and death, emphasize the urgent need to explore alternative strategies to manage P. aeruginosa infections. Enhance understanding of the antimicrobial mechanisms of the Surfactant Protein A, and the mechanisms by which P. aeruginosa confers resistance/susceptibility to Surfactant Protein A may lead to new treatment strategies for life-threatening pneumonias.